Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application
| Autor: | Anne Galy, Maria Antonietta Zanta-Boussif, Muriel Audit, Otto Wilhelm Merten, Marina Radrizzani, Christine Jenny, Sylvain Fauchille, Patricia Noguiez-Hellin, Céline Dugué, Luigi Naldini, Nicolas Laroudie, Hélène Chautard, Giuliana Vallanti, Sabine Charrier |
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| Přispěvatelé: | Merten, Ow, Charrier, S, Laroudie, N, Fauchille, S, Dugue, C, Jenny, C, Audit, M, Zanta Boussif, Ma, Chautard, H, Radrizzani, M, Vallanti, G, Naldini, Luigi, Noguiez Hellin, P, Galy, A., Immunologie moléculaire et biothérapies innovantes (IMBI), Généthon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université d'Évry-Val-d'Essonne (UEVE)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Laboratoire Environnements Sédimentaires - Géosciences Marines (GM/LES), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Centre de biophysique moléculaire (CBM), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Laboratoire Environnements Sédimentaires (LES), Géosciences Marines (GM), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER)-Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Université d'Orléans (UO)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2010 |
| Předmět: |
Lentivirus/*genetics/physiology
Drug Contamination/legislation & Wiskott-Aldrich Syndrome/therapy [SDV]Life Sciences [q-bio] Genetic enhancement Cell Culture Techniques Industrial Microbiology/*methods Transduction (genetics) 0302 clinical medicine Proviruses Transduction Genetic Gene Order Hematopoietic Stem Cells/metabolism Genetic Vectors/*biosynthesis/*genetics/physiology Transgenes Plasmids/genetics 0303 health sciences biology Wiskott-Aldrich Syndrome Titer Vesicular stomatitis virus 030220 oncology & carcinogenesis Molecular Medicine Drug Contamination Plasmids Quality Control Transgenes/genetics Genetic Vectors Virus Viral vector Cell Line Transduction 03 medical and health sciences Industrial Microbiology Genetic jurisprudence/prevention & Genetics Humans Molecular Biology Proviruses/genetics 030304 developmental biology Genetic Therapy Lentivirus biology.organism_classification Hematopoietic Stem Cells Virology HEK293 Cells Gene Expression Regulation Cell culture control Ex vivo |
| Zdroj: | Human Gene Therapy Human Gene Therapy, Mary Ann Liebert, 2011, 22 (3), pp.343-56. ⟨10.1089/hum.2010.060⟩ Human Gene Therapy, 2011, 22 (3), pp.343-56. ⟨10.1089/hum.2010.060⟩ |
| ISSN: | 1557-7422 1043-0342 |
| DOI: | 10.1089/hum.2010.060⟩ |
| Popis: | International audience; From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 x 10(9) infectious particles per milliliter were obtained, generating up to 6 x 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications. |
| Databáze: | OpenAIRE |
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